Compositions and methods for treatment of mitochondrial diseases

ABSTRACT

Compounds, compositions, and methods are provided for treatment of disorders related to mitochondrial dysfunction. The methods comprise administering to a mammal a composition containing pyrimidine nucleotide precursors in amounts sufficient to treat symptoms resulting from mitochondrial respiratory chain deficiencies.

FIELD OF THE INVENTION

This invention relates generally to compounds and methods for treatmentand prevention of diseases, developmental delays, and symptoms relatedto mitochondrial dysfunction. Pyrimidine nucleotide precursors areadministered to a mammal, including a human, for the purpose ofcompensating for mitochondrial dysfunction and for improvingmitochondrial functions.

BACKGROUND OF THE INVENTION

Mitochondria are cellular organelles present in most eukaryotic cells.One of their primary functions is oxidative phosphorylation, a processthrough which energy derived from metabolism of fuels like glucose orfatty acids is converted to ATP, which is then used to drive variousenergy-requiring biosynthetic reactions and other metabolic activities.Mitochondria have their own genomes, separate from nuclear DNA,comprising rings of DNA with about 16,000 base pairs in human cells.Each mitochondrion may have multiple copies of its genome, andindividual cells may have hundreds of mitochondria.

Mitochondrial dysfunction contributes to various disease states. Somemitochondrial diseases are due to mutations or deletions in themitochondrial genome. Mitochondria divide and proliferate with a fasterturnover rate than their host cells, and their replication is undercontrol of the nuclear genome. If a threshold proportion of mitochondriain a cell is defective, and if a threshold proportion of such cellswithin a tissue have defective mitochondria, symptoms of tissue or organdysfunction can result. Practically any tissue can be affected, and alarge variety of symptoms may be present, depending on the extent towhich different tissues are involved.

A fertilized ovum might contain both normal and genetically defectivemitochondria. The segregation of defective mitochondria into differenttissues during division of this ovum is a stochastic process, as will bethe ratio of defective to normal mitochondria within a given tissue orcell (although there can be positive or negative selection for defectivemitochondrial genomes during mitochondrial turnover within cells). Thus,a variety of different pathologic phenotypes can emerge out of aparticular point mutation in mitochondrial DNA. Conversely, similarphenotypes can emerge from mutations or deletions affecting differentgenes within mitochondrial DNA. Clinical symptoms in congenitalmitochondrial diseases often manifest in postmitotic tissues with highenergy demands like brain, muscle, optic nerve, and myocardium, butother tissues including endocrine glands, liver, gastrointestinal tract,kidney, and hematopoietic tissue are also involved, again depending inpart on the segregation of mitochondria during development, and on thedynamics of mitochondrial turnover over time.

In addition to congenital disorders involving inherited defectivemitochondria, acquired mitochondrial dysfunction contributes todiseases, particularly neurodegenerative disorders associated with aginglike Parkinson's, Alzheirner's, Huntington's Diseases. The incidence ofsomatic mutations in mitochondrial DNA rises exponentially with age;diminished respiratory chain activity is found universally in agingpeople. Mitochondrial dysfunction is also implicated in excitotoxicneuronal injury, such as that associated with seizures or ischemia.

Treatment of diseases involving mitochondrial dysfunction has heretoforeinvolved administration of vitamins and cofactors used by particularelements of the mitochondrial respiratory chain. Coenzyme Q(ubiquinone), nicotinamide, riboflavin, carnitine, biotin, and lipoicacid are used in patients with mitochondrial disease, with occasionalbenefit, especially in disorders directly stemming from primarydeficiencies of one of these cofactors. However, while useful inisolated cases, no such metabolic cofactors or vitamins have been shownto have general utility in clinical practice in treating mitochondrialdiseases. Similarly, dichloracetic acid (DCA) has been used to treatmitochondrial cytopathies such as MELAS; DCA inhibits lactate formationand is primarily useful in cases of mitochondrial diseases whereexcessive lactate accumulation itself is contributing to symptoms.However, DCA does not address symptoms related to mitochondrialinsufficiency per se and can be toxic to some patients, depending on theunderlying molecular defects.

Mitochondrial diseases comprise disorders caused by a huge variety ofmolecular lesions or defects, with the phenotypic expression of diseasefurther complicated by stochastic distributions of defectivemitochondria in different tissues.

Commonly owned U.S. Pat. No. 5,583,117 discloses acylated derivatives ofcytidine and uridine. Commonly owned application PCT/US 96/10067discloses the use of acylated pyrimidine nucleosides to reduce thetoxicity of chemotherapeutic and antiviral pyrimidine nucleosideanalogs.

OBJECTS OF THE INVENTION

It is an object of the invention to provide compositions and methods fortreating disorders or pathophysiology associated with mitochondrialdysfunction or mitochondrial respiratory chain dysfunction in a mammal,including a human.

It is an object of the invention to provide compounds and compositionsthat improve tissue resistance to mitochondrial dysfunction in vivo.

It is an object of the invention to provide compositions and methods fortreatment of mitochondrial diseases.

It is an object of the invention to provide agents which compensatebroadly for mitochondrial deficits involving a wide variety of molecularpathologies, since, in many cases, precise diagnosis of molecularlesions in mitochondrial disorders is difficult.

It is an object of the invention to provide a practical treatment formitochondrial diseases that is beneficial in the case of mitochondrialelectron transport chain deficits regardless of the specific moleculardefects.

It is an object of the invention to provide not only for the relativelyrare congenital diseases related to mitochondrial DNA defects, but alsofor significant neuromuscular and neurodevelopmental disorders thatappear in childhood and for common age-related degenerative diseaseslike Alzheimer's or Parkinson's Diseases.

It is an object of the invention to provide compositions and methods fortreatment and prevention of neurodegenerative and neuromusculardisorders.

It is an object of the invention to provide compositions and methods fortreatment and prevention of epilepsy.

It is an object of the invention to provide compositions and methods fortreatment and prevention of migraine.

It is an object of the invention to provide compositions and methods forpreventing death or dysfunction of postmitotic cells in a mammal,including a human.

It is an object of the invention to provide compositions and methods fortreatment of neurodevelopmental delay disorders

It is a further object of the invention to provide a composition fortreatment or prevention of tissue damage due to hypoxia or ischemia.

It is a further object of this invention to provide compositions andmethods for treating or preventing ovarian dysfunction, menopause, orsecondary consequences of menopause.

It is a further object of the invention to provide compositions andmethods for reducing side effects of cancer chemotherapies due tochemotherapy-induced mitochondrial injury.

It is a further object of the invention to provide a method fordiagnosing mitochondrial disease and dysfunction.

SUMMARY OF THE INVENTION

The subject invention provides a method for treating pathophysiologicalconsequences of mitochondrial respiratory chain deficiency in a mammalcomprising administering to such a mammal in need of such treatment anamount of a pyrimidine nucleotide precursor effective in reducing thepathophysiological consequences. Additionally, the invention provides amethod of preventing pathophysiological consequences of mitochondrialrespiratory chain deficiency comprising administering to a mammal anamount of a pyrimidine nucleotide precursor effective in preventing thepathophysiological consequences.

In mitochondrial disease the compounds and compositions of the inventionare useful for attenuating clinical sequelae stemming from respiratorychain deficiencies. Respiratory chain deficiencies underlyingmitochondrial disease are caused by various factors including congenitalor inherited mutations and deletions in mitochondrial DNA, deficits innuclear-encoded proteins affecting respiratory chain activity, as wellas somatic mutations, elevated intracellular calcium, excitotoxicity,nitric oxide, hypoxia and axonal transport defects.

The subject invention provides compounds, compositions, and methods forpreventing or reducing death and dysfunction of postmitotic cellsbearing mitochondrial respiratory chain deficits.

The subject invention furthermore provides compounds, compositions, andmethods for treating neurodevelopmental delays in language, motor,executive function, cognitive, and neuropsychological social skills.

The subject invention also relates to treatment of disorders andconditions that are herein disclosed as conditions to whichmitochondrial defects contribute and which therefore are subject totreatment with compounds, and compositions of the invention. Theseinclude side effects of cancer chemotherapy like peripheralneuropathies, nephropathies, fatigue, and early menopause, as well asovulatory abnormalities and normal menopause itself.

The subject invention also relates to a method for diagnosingmitochondrial diseases by treating patients with a pyrimidine nucleotideprecursor and assessing clinical benefit in selected signs and symptoms.

The invention, as well as other objects, features and advantages thereofwill be understood more clearly and fully from the following detaileddescription, when read with reference to the accompanying results of theexperiments discussed in the examples below.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention is related to compounds, compositions, and methodsfor treating or preventing a variety of clinical disorders secondary tomitochondrial dysfunction, especially deficits in the activity ofcomponents of the mitochondrial respiratory chain. Such disordersinclude congenital mitochondrial cytopathies, neurodevelopmental delays,age-related neurodegenerative diseases, as well as particular diseasesaffecting the heart, peripheral and autonomic nerves, skeletal muscle,pancreas and other tissues and organs.

A. Definitions

“Mitochondrial disease” refers to disorders to which deficits inmitochondrial respiratory chain activity contribute in the developmentof pathophysiology of such disorders in a mammal. This categoryincludes 1) congenital genetic deficiencies in activity of one or morecomponents of the mitochondrial respiratory chain; 2) acquireddeficiencies in the activity of one or more components of themitochondrial respiratory chain, wherein such deficiencies are causedby, inter alia, a) oxidative damage during aging; b) elevatedintracellular calcium; c) exposure of affected cells to nitric oxide; d)hypoxia or ischemia; or e) microtubule-associated deficits in axonaltransport of mitochondria.

The mitochondrial respiratory chain (also known as the electrontransport chain) comprises 5 major complexes: Complex I NADH: ubiquinonereductase Complex II Succinate: ubiquinone reductase Complex IIIubiquinol: cytochrome-c reductase Complex IV cytochrome-c oxidaseComplex V ATP synthase

Complexes I and II accomplish the transfer of electrons from metabolicfuels like glycolysis products and fatty acids to ubiquinone (CoenzymeQ), converting it to ubiquinol. Ubiquinol is converted back toubiquinone by transfer of electrons to cytochrome c in Complex III.Cytochrome c is reoxidized at Complex IV by transfer of electrons tomolecular oxygen, producing water. Complex V utilizes potential energyfrom the proton gradient produced across the mitochondrial membrane bythese electron transfers into ATP.

Dihydro-orotate dehydrogenase (DHODH), is an enzyme involved in de novosynthesis of uridine nucleotides. DHODH activity is coupled to therespiratory chain via transfer of electrons from dihydro-orotate toubiquinone; these electrons are then passed on to cytochrome c andoxygen via Complexes III and IV respectively. Only Complexes III and IVare directly involved in pyrimidine biosynthesis. Orotate produced bythe action of DHODH is converted to uridine monophosphate byphosphoribosylation and decarboxylation.

“Pyrimidine nucleotide precursors” in the context of the invention areintermediates in either the de novo or salvage pathways of pyrimidinenucleotide synthesis that enter into pyrimidine synthesis either distalto DHODH (e.g. orotate) or which do not require DHODH activity forconversion to pyrimidine nucleotides (e.g. cytidine, uridine, or acylderivatives of cytidine or uridine). Also included within the scope ofthe invention are pyrimidine nucleoside phosphates (e.g. nucleotides,cytidine diphosphocholine, uridine diphosphoglucose); these compoundsare degraded to the level of uridine or cytidine prior to entry intocells and anabolism. Acyl derivatives of cytidine and uridine havebetter oral bioavailability than the parent nucleosides or nucleotides.Orotic acid and esters thereof are converted to uridine nucleotides andare also useful for accomplishing the goals of the invention.

B. Compounds of the Invention

A primary feature of the present invention is the unexpected discoverythat administration of pyrimidine nucleotide precursors is effective intreatment of a large variety of symptoms and disease states related tomitochondrial dysfunction.

Tissue pyrimidine nucleotide levels are increased by administration ofany of several precursors. Uridine and cytidine are incorporated intocellular nucleotide pools by phosphorylation at the 5′ position;cytidine and uridine nucleotides are interconvertible through enzymaticamination and de-amination reactions. Orotic acid is a key intermediatein de novo biosynthesis of pyrimidine nucleotides. Incorporation oforotic acid into nucleotide pools requires cellular phosphoribosylpyrophosphate (PRPP). Alternatively (or in addition to provision ofexogenous nucleotide precursors), availability of uridine to tissues isincreased by administration of compounds which inhibit uridinephosphorylase, the first enzyme in the pathway for degradation ofuridine. The compounds of the invention useful in treating mitochondrialdiseases and related disorders include uridine, cytidine, orotate,orally bioavailable acyl derivatives or esters of these pyrimidinenucleotide precursors, and inhibitors of the enzyme uridinephosphorylase.

In reference to acyl derivatives of cytidine and uridine, the followingdefinitions pertain:

The term “acyl derivative” as used herein means a derivative of apyrimidine nucleoside in which a substantially nontoxic organic acylsubstituent derived from a carboxylic acid is attached to one or more ofthe free hydroxyl groups of the ribose moiety of the oxypurinenucleoside with an ester linkage and/or where such a substituent isattached to the amine substituent on the purine ring of cytidine, withan amide linkage. Such acyl substituents are derived from carboxylicacids which include, but are not limited to, compounds selected from thegroup consisting of a fatty acid, an amino acid, nicotinic acid,dicarboxylic acids, lactic acid, p-aminobenzoic acid and orotic acid.Advantageous acyl substituents are compounds which are normally presentin the body, either as dietary constituents or as intermediarymetabolites.

The term “pharmaceutically acceptable salts” as used herein means saltswith pharmaceutically acceptable acid or base addition salts of thederivatives, which include, but are not limited to, sulfuric,hydrochloric, or phosphoric acids, or, in the case of orotate, sodium orcalcium hydroxides, and cationic amino acids, especially lysine.

The term “amino acids” as used herein includes, but is not limited to,glycine, the L forms of alanine, valine, leucine, isoleucine,phenylalanine, tyrosine, proline, hydroxyproline, serine, threonine,cysteine, cystine, methionine, tryptophan, aspartic acid, glutamic acid,arginine, lysine, histidine, ornithine, hydroxylysine, carnitine, andother naturally occurring amino acids.

The term “fatty acids” as used herein means aliphatic carboxylic acidshaving 2-22 carbon atoms. Such fatty acids may be saturated, partiallysaturated or polyunsaturated.

The term “dicarboxylic acids” as used herein means fatty acids with asecond carboxylic acid substituent.

Compounds of the invention have the following structures:

In all cases except where indicated, letters and letters with subscriptssymbolizing variable substituents in the chemical structures of thecompounds of the invention are applicable only to the structureimmediately preceding the description of the symbol.

(1) An acyl derivative of uridine having the formula:

wherein R₁, R₂, R₃ and R₄ are the same or different and each is hydrogenor an acyl radical of a metabolite, provided that at least one of said Rsubstituents is not hydrogen, or a pharmaceutically acceptable saltthereof.

(2) An acyl derivative of cytidine having the formula:

wherein R₁, R₂, R₃ and R₄ are the same or different and each is hydrogenor an acyl radical of a metabolite, provided that at least one of said Rsubstituents is not hydrogen, or a pharmaceutically acceptable saltthereof.

The compounds of the invention useful in treating mitochondrial diseasesinclude:

(3) An acyl derivative of uridine having the formula:

wherein R₁, R₂, and R₃ are the same, or different, and each is hydrogenor an acyl radical of

a. an unbranched fatty acid with 2 to 22 carbon atoms,

b. an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine and ornithine,

c. a dicarboxylic acid having 3-22 carbon atoms,

d. a carboxylic acid selected from one or more of the group consistingof glycolic acid, pyruvic acid, lactic acid, enolpyruvic acid, lipoicacid, pantothenic acid, acetoacetic acid, p-aminobenzoic acid,betahydroxybutyric acid, orotic acid, and creatine.

(4) An acyl derivatives of cytidine having the formula:

wherein R₁, R₂, R₃, and R₄ are the same, or different, and each ishydrogen or an acyl radical of

a. an unbranched fatty acid with 2 to 22 carbon atoms,

b. an amino acid selected from the group consisting of glycine, the Lforms of phenylalanine, alanine, valine, leucine, isoleucine, tyrosine,proline, hydroxyproline, serine, threonine, cystine, cysteine, asparticacid, glutamic acid, arginine, lysine, histidine carnitine andornithine,

c. a dicarboxylic acid having 3-22 carbon atoms,

d. a carboxylic acid selected from one or more of the group consistingof glycolic acid, pyruvic acid, lactic acid, enolpyruvic acid, lipoicacid, pantothenic acid, acetoacetic acid, p-aminobenzoic acid,betahydroxybutyric acid, orotic acid, and creatine.

(5) An acyl derivative of uridine having the formula:

wherein at least one of R₁, R₂, or R₃ is a hydrocarbyloxycarbonyl moietycontaining 2-26 carbon atoms and the remaining R substituents areindependently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety orH or phosphate.

(6) An acyl derivative of cytidine having the formula:

wherein at least one of R₁, R₂, R₃ or R₄ is a hydrocarbyloxycarbonylmoiety containing 2-26 carbon atoms and the remaining R substituents areindependently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety orH or phosphate.

(7) Orotic acid or salts thereof:

Pharmaceutically-acceptable salts of orotic acid include those in whichthe cationic component of the salt is sodium, potassium, a basic aminoacid such as arginine or lysine, methylglucamine, choline, or any othersubstantially nontoxic water soluble cation with a molecular weight lessthan about 1000 daltons.

8) Alcohol-substituted orotate derivatives:

wherein R₁ is a radical of an alcohol containing 1 to 20 carbon atomsjoined to orotate via an ester linkage.

Also encompassed by the invention are the pharmaceutically acceptablesalts of the above-noted compounds.

Advantageous compounds of the invention are short-chain (2 to 6 carbonatoms) fatty acid esters of uridine or cytidine. Particularlyadvantageous compounds are triacetyluridine or triacetylcytidine. Suchcompounds have better oral bioavailabilty than the parent nucleosides,and are rapidly deacetylated following absorption after oraladministration.

Pyruvic acid is useful for treatment of cells with defectivemitochondrial function. Cells with reduced capability for mitochondrialoxidative phosphorylation must rely on glycolysis for generation of ATP.Glycolysis is regulated by the redox state of cells. Specifically, NAD+is required for optimal glucose flux, producing NADH in the process. Inorder to maximize energy production from glycolysis, NADH must bereoxidized to NAD+. Exogenous pyruvate can reoxidize NADH, in part via aplasma membrane enzyme, NADH Oxidase.

Uridine tripyruvate (2′,3′,5′-tri-O-pyruvyluridine) provides thebenefits of both pyrimidines and pyruvate, delivering both with a singlechemical entity, and avoiding the load of sodium, calcium, or othercations in the corresponding salts of pyruvic acid.

Inhibitors of Uridine Phosphorylase

An alternative or complementary strategy for treating mitochondrialdiseases involves inhibition of uridine catabolism with an inhibitor ofthe enzyme uridine phosphorylase.

Examples of inhibitors of uridine phosphorylase that are useful fortreatment of mitochondrial disease include but are not limited to5-benzyl barbiturate or 5-benzylidene barbiturate derivatives including5-benzyl barbiturate, 5-benzyloxybenzyl barbiturate,5-benzyloxybenzyl-1-[(1-hydroxy-2-ethoxy)methyl]barbiturate,5-benzyloxybenzylacetyl-1-[(1-hydroxy-2-ethoxy)methyl]barbiturate, and5-methoxybenzylacetylacyclobarbiturate, 2,2′-anhydro-5-ethyluridine,5-ethyl-2-deoxyuridine and acyclouridine compounds, particularly5-benzyl substituted acyclouridine congeners including but not limitedto benzylacyclouridine, benzyloxybenzylacyclouridine,aminomethyl-benzylacyclouridine,aminomethylbenzyloxy-benzylacyclouridine,hydroxymethyl-benzylacyclouridine, andhydroxymethyl-benzyloxy-benzylacyclouridine. See also WO 89/09603 and WO91/16315, hereby incorporated by reference.

C. Compositions of the Invention

In one embodiment of the invention, novel pharmaceutical compositionscomprise as an active agent one or more pyrimidine nucleotide precursorsselected from the group consisting of uridine, cytidine, orotic acid orits salts or esters, and acyl derivatives of these pyrimidine nucleotideprecursors, together with a pharmaceutically acceptable carrier.

The compositions, depending on the intended use and route ofadministration, are manufactured in the form of a liquid, a suspension,a tablet, a capsule, a dragee, an injectable solution, or a suppository(see discussion of formulation below).

In another embodiment of the invention, the composition comprises atleast one pyrimidine nucleotide precursor and an agent which inhibitsthe degradation of uridine, such as an inhibitor of the enzyme uridinephosphorylase. Examples of inhibitors of uridine phosphorylase includebut are not limited to 5-benzyl barbiturate or 5-benzylidene barbituratederivatives including 5-benzyl barbiturate, 5-benzyloxybenzylbarbiturate,5-benzyloxybenzyl-1-[(1-hydroxy-2-ethoxy)methyl]barbiturate,5-benzyloxybenzylacetyl-1-[(1-hydroxy-2-ethoxy)methyl]barbiturate, and5-methoxybenzylacetylacyclobarbiturate, 2,2′-anhydro-5-ethyluridine, andacyclouridine compounds, particularly 5-benzyl substituted acyclouridinecongeners including but not limited to benzylacyclouridine,benzyloxybenzyl-acyclouridine, aminomethyl-benzylacyclouridine,aminomethylbenzyloxybenzylacyclouridine,hydroxymethyl-benzylacyclouridine, andhydroxymethyl-benzyloxybenzylacyclouridine. Furthermore, it is withinthe scope of the invention to utilize an inhibitor of uridinephosphorylase alone, without coadministration of a pyrimidine nucleotideprecursor, for the purpose of treating mitochondrial diseases orpathophysiologies associated with mitochondrial respiratory chaindysfunction.

Further embodiments of the invention comprise a pyrimidine nucleotideprecursor combined with one or more other agents with protective orsupportive activity relative to mitochondrial structure and function.Such agents, presented with recommended daily doses in mitochondrialdiseases include, but are not limited to, pyruvate (1 to 10 grams/day).Coenzyme Q (1 to 4 mg/kg/day), alanine (1-10 grams/day), lipoic acid (1to 10 mg/kg/day), carnitine (10 to 100 mg/kg/day), riboflavin (20 to 100mg/day, biotin (1 to 10 mg/day), nicotinamide (20 to 100 mg/day), niacin(20 to 100 mg/day), Vitamin C (100 to 1000 mg/day), Vitamin E (200-400mg/day), and dichloroacetic acid or its salts. In the case of pyruvate,this active agent can be administered as pyruvic acid, pharmaceuticallyacceptable salts thereof, or pyruvic acid esters having an alcoholmoiety containing 2 to 10 carbon atoms.

D. Therapeutic Uses of the Compounds and Compositions of the Invention

Diseases related to mitochondrial respiratory chain dysfunction can bedivided into several categories based on the origin of mitochondrialdefects.

Congenital mitochondrial diseases are those related to hereditarymutations, deletions, or other defects in mitochondrial DNA or innuclear genes regulating mitochondrial DNA integrity, or in nucleargenes encoding proteins that are critical for mitochondrial respiratorychain function.

Acquired mitochondrial defects comprise primarily 1) damage tomitochondrial DNA due to oxidative processes or aging; 2) mitochondrialdysfunction due to excessive intracellular and intramitochondrialcalcium accumulation; 3) inhibition of respiratory chain complexes withendogenous or exogenous respiratory chain inhibitors; 4) acute orchronic oxygen deficiency; and 5) impaired nuclear-mitochondrialinteractions, e.g. impaired shuttling of mitochondria in long axons dueto microtubule defects.

The most fundamental mechanisms involved in acquired mitochondrialdefects, and which underlie pathogenesis of a variety of forms of organand tissue dysfunction, include:

Calcium accumulation: A fundamental mechanism of cell injury, especiallyin excitable tissues, involves excessive calcium entry into cells, as aresult of either leakage through the plasma membrane or defects inintracellular calcium handling mechanisms. Mitochondria are major sitesof calcium sequestration, and preferentially utilize energy from therespiratory chain for taking up calcium rather than for ATP synthesis,which results in a downward spiral of mitochondrial failure, sincecalcium uptake into mitochondria results in diminished capabilities forenergy transduction.

Excitotoxicity: Excessive stimulation of neurons with excitatory aminoacids is a common mechanism of cell death or injury in the centralnervous system. Activation of glutamate receptors, especially of thesubtype designated NMDA receptors, results in mitochondrial dysfunction,in part through elevation of intracellular calcium during excitotoxicstimulation. Conversely, deficits in mitochondrial respiration andoxidative phosphorylation sensitizes cells to excitotoxic stimuli,resulting in cell death or injury during exposure to levels ofexcitotoxic neurotransmitters or toxins that would be innocuous tonormal cells.

Nitric oxide exposure: Nitric oxide (−1 micromolar) inhibits cytochromeoxidase (Complex IV) and thereby inhibits mitochondrial respiration(Brown G C, Mol. Cell. Biochem. 174:189-192, 1997); moreover, prolongedexposure to NO irreversibly reduces Complex I activity. Physiological orpathophysiological concentrations of NO thereby inhibit pyrimidinebiosynthesis. Nitric oxide is implicated in a variety ofneurodegenerative disorders, and is involved in mediation of excitotoxicand post-hypoxic damage to neurons.

Hypoxia: Oxygen is the terminal electron acceptor in the respiratorychain. Oxygen deficiency impairs electron transport chain activity,resulting in diminished pyrimidine synthesis as well as diminished ATPsynthesis via oxidative phosphorylation. Human cells proliferate andretain viability under virtually anaerobic conditions if provided withuridine and pyruvate (or a similarly effective agent for oxidizing NADHto optimize glycolytic ATP production).

Nuclear-mitochondrial interactions: Transcription of mitochondrial DNAencoding respiratory chain components requires nuclear factors. Inneuronal axons, mitochondria must shuttle back and forth to the nucleusin order to maintain respiratory chain activity. If axonal transport isimpaired by hypoxia or by drugs like taxol which affect microtubulestability, mitochondria distant from the nucleus undergo loss ofcytochrome oxidase activity.

In the nervous system especially, mitochondrial respiratory chaindeficits have two generalizable consequences: 1) Delayed or aberrantdevelopment of neuronal circuits within the nervous system; and 2)accelerated degeneration of neurons and neural circuits, either acutelyor over a period of years, depending on the severity of themitochondrial deficits and other precipitating factors. Analogouspatterns of impaired development and accelerated degeneration pertain tonon-neural tissues and systems as well.

Mitochondrial Dysfunction and Pyrimidine Biosynthesis

Cells with severely damaged mitochondria (including total deletion ofmitochondrial DNA, with a consequent shutdown of respiratory chainactivity) can survive in culture if provided with two agents whichcompensate for critical mitochondrial functions: uridine and pyruvate.Uridine is required in vitro because a limiting enzyme for de novosynthesis of uridine nucleotides, dihydro-orotate dehydrogenase (DHODH),is coupled to the mitochondrial respiratory chain, via ubiquinone as aproximal electron acceptor, cytochrome c as an intermediate, and oxygenas a terminal electron acceptor (Loffler et al., Mol. Cell. Biochem.174:125-129, 1997). DHODH is required for synthesis of orotate, which isthen phosphoribosylated and decarboxylated to produce uridinemonophosphate (UMP). All other pyrimidines in cells are derived fromUMP. Cells from patients with mitochondrial disease due to defects inmitochondrial DNA require exogenous uridine in order to survive outsideof the milieu of the body, wherein pyrimidines, derived from other cellsor the diet, and transported via the circulation, are prima faciesufficient to support their viability (Bourgeron, et al. Neuromusc.Disord. 3:605-608, 1993). Significantly, intentional inhibition of DHODHwith drugs like Brequinar or Leflunomide results in dose-limitingcytotoxic damage to the hematopoietic system and gastrointestinalmucosa, in contrast to the predominant involvement of postmitotictissues like the nervous system and muscle in clinical mitochondrialdisease.

An important feature of the subject invention is the unexpected resultthat treatment of patients with mitochondrial disease caused by avariety of underlying molecular defects results in clinical improvementin a diverse assortment of symptoms in vivo in patients (Examples 1-4).It is significant and further unexpected that clinical benefit has beenobserved in patients with no detectable defects in the respiratory chaincomplexes (III and IV) that are involved in pyrimidine biosynthesis.

Treatment of Congenital Mitochondrial Cytopathies

Mitochondrial DNA Defects

A number of clinical syndromes have been linked to mutations ordeletions in mitochondrial DNA. Mitochondrial DNA is inheritedmaternally, with virtually all of the mitochondria in the body derivedfrom those provided by the oocyte. If there is a mixture of defectiveand normal mitochondria in an oocyte, the distribution and segregationof mitochondria is a stochastic process. Thus, mitochondrial diseasesare often multisystem disorders, and a particular point mutation inmitochondrial DNA, for example, can result in dissimilar sets of signsand symptoms in different patients. Conversely, mutations in twodifferent genes in mitochondrial DNA can result in similar symptomcomplexes.

Nonetheless, some consistent symptom patterns have emerged inconjunction with identified mitochondrial DNA defects, and thesecomprise the classic “mitochondrial diseases”, some of which are listedimmediately below. Nonetheless, an important aspect of the subjectinvention is the recognition that the concept of mitochondrial diseaseand its treatment with compounds and compositions of the inventionextends to many other disease conditions which are also disclosedherein.

Some of the major mitochondrial diseases associated with mutations ordeletions of mitochondrial DNA include:

MELAS: (Mitochondrial Encephalomyopathy Lactic Acidemia, and Stroke-likeepisodes.

MERRF: Myoclonic Epilepsy with “Ragged Red” (muscle) Fibers

NARP: Neurogenic muscle weakness, Ataxia and Retinitis Pigmentosa

LHON: Leber's Hereditary Optic Neuropathy

Leigh's Syndrome (Subacute Necrotizing Encephalomyopathy)

PEO: Progressive External Opthalmoplegia

Kearns-Sayres Syndrome (PEO, pigmentary retinopathy, ataxia, andheart-block)

Other common symptoms of mitochondrial diseases which may be presentalone or in conjunction with these syndromes include cardiomyopathy,muscle weakness and atrophy, developmental delays (involving motor,language, cognitive or executive function), ataxia, epilepsy, renaltubular acidosis, peripheral neuropathy, optic neuropathy, autonomicneuropathy, neurogenic bowel dysfunction, sensorineural deafness,neurogenic bladder dysfunction, dilating cardiomyopathy, migraine,hepatic failure, lactic acidemia, and diabetes mellitus.

In addition, gene products and tRNA encoded by mitochondrial DNA, manyproteins involved in, or affecting, mitochondrial respiration andoxidative phosphorylation are encoded by nuclear DNA. In fact,approximately 3000 proteins, or 20% of all proteins encoded by thenuclear genome, are physically incorporated into, or associated with,mitochondria and mitochondrial functions, although only about 100 aredirectly involved as structural components of the respiratory chain.Therefore, mitochondrial diseases involve not only gene products ofmitochondrial DNA, but also nuclear encoded proteins affectingrespiratory chain function.

Metabolic stressors like infections can unmask mitochondrial defectsthat do not necessarily yield symptoms under normal conditions.Neuromuscular or neurological setbacks during infection are a hallmarkof mitochondrial disease. Conversely, mitochondrial respiratory chaindysfunction can render cells vulnerable to stressors that wouldotherwise be innocuous.

As is demonstrated in the Examples, compounds and compositions of theinvention are useful for treatment of a very broad spectrum of signs andsymptoms in mitochondrial diseases with different underlying molecularpathologies. The broad applicability of the methods of the invention areunexpected and set the compounds and compositions of the invention apartfrom other therapies of mitochondrial disease that have been attemptede.g. Coenzyme Q, B vitamins, carnitine, and lipoic acid, which generallyaddress very specific reactions and cofactors involved in mitochondrialfunction and which are therefore useful only in isolated cases. However,such metabolic interventions with antioxidants and cofactors ofrespiratory chain complexes are compatible with concurrent treatmentwith compounds and compositions of the invention, and in fact are usedto their best advantage in combination with compounds and compositionsof the invention.

Treatment of Neuromuscular Degenerative Disorders

Friedreich's Ataxia

A gene defect underlying Friedreich's Ataxia (FA), the most commonhereditary ataxia, was recently identified and is designated “frataxin”.In FA, after a period of normal development, deficits in coordinationdevelop which progress to paralysis and death, typically between theages of 30 and 40. The tissues affected most severely are the spinalcord, peripheral nerves, myocardium, and pancreas. Patients typicallylose motor control and are confined to wheel chairs, and are commonlyafflicted with heart failure and diabetes.

The genetic basis for FA involves GAA trinucleotide repeats in an intronregion of the gene encoding frataxin. The presence of these repeatsresults in reduced transcription and expression of the gene. Frataxin isinvolved in regulation of mitochondrial iron content. When cellularfrataxin content is subnormal, excess iron accumulates in mitochondria,promoting oxidative damage and consequent mitochondrial degeneration anddysfunction.

When intermediate numbers of GAA repeats are present in the frataxingene intron, the severe clinical phenotype of ataxia may not develop.However, these intermediate-length trinucleotide extensions are found in25 to 30% of patients with non-insulin dependent diabetes mellitus,compared to about 5% of the nondiabetic population.

Compounds and compositions of the invention are useful for treatingpatients with disorders related to deficiencies or defects in frataxin,including Friedreich's Ataxia, myocardial dysfunction, diabetes mellitusand complications of diabetes like peripheral neuropathy. Conversely,diagnostic tests for presumed frataxin deficiencies involving PCR testsfor GAA intron repeats are useful for identifying patients who willbenefit from treatment with compounds and compositions of the invention.

Muscular Dystrophy

Muscular dystrophy refers to a family of diseases involvingdeterioration of neuromuscular structure and function, often resultingin atrophy of skeletal muscle and myocardial dysfunction. In the case ofDuchenne muscular dystrophy, mutations or deficits in a specificprotein, dystrophin, are implicated in its etiology. Mice with theirdystrophin genes inactivated display some characteristics of musculardystrophy, and have an approximately 50% deficit in mitochondrialrespiratory chain activity. A final common pathway for neuromusculardegeneration in most cases is calcium-mediated impairment ofmitochondrial function. Compounds and compositions of the invention areuseful for reducing the rate of decline in muscular functionalcapacities and for improving muscular functional status in patients withmuscular dystrophy.

Multiple Sclerosis

Multiple sclerosis (MS) is a neuromuscular disease characterized byfocal inflammatory and autoimmune degeneration of cerebral white matter.Periodic exacerbations or attacks are significantly correlated withupper respiratory tract and other infections, both bacterial and viral,indicating that mitochondrial dysfunction plays a role in MS. Nitricoxide Depression of neuronal mitochondrial respiratory chain activitycaused by Nitric Oxide (produced by astrocytes) is implicated as amolecular mechanism contributing to MS.

Compounds and compositions of the invention are useful for treatment ofpatients with multiple sclerosis, both prophylactically and duringepisodes of disease exacerbation.

Treatment of Disorders of Neuronal Instability

Treatment of Seizure Disorders

Epilepsy is often present in patients with mitochondrial cytopathies,involving a range of seizure severity and frequency, e.g. absence,tonic, atonic, myoclonic, and status epilepticus, occurring in isolatedepisodes or many times daily.

In patients with seizures secondary to mitochondrial dysfunction,compounds and methods of the invention are useful for reducing frequencyand severity of seizure activity.

Treatment and Prevention of Migraine

Metabolic studies on patients with recurrent migraine headaches indicatethat deficits in mitochondrial activity are commonly associated withthis disorder, manifesting as impaired oxidative phosphorylation andexcess lactate production. Such deficits are not necessarily due togenetic defects in mitochondrial DNA. Migraineurs are hypersensitive tonitric oxide, an endogenous inhibitor of Cytochrome c Oxidase. Inaddition, patients with mitochondrial cytopathies, e.g. MELAS, oftenhave recurrent migraines.

In patients with recurrent migraine headaches, compounds, compositions,and methods of the invention are useful for prevention and treatment,especially in the case of headaches refractory to ergot compounds orserotonin receptor antagonists.

As demonstrated in Example 1, compounds and compositions of theinvention are useful for treatment of migraines associate withmitochondrial dysfunction.

Treatment of Developmental Delay

Delays in neurological or neuropsychological development are often foundin children with mitochondrial diseases. Development and remodeling ofneural connections requires intensive biosynthetic activity,particularly involving synthesis of neuronal membranes and myelin, bothof which require pyrimidine nucleotides as cofactors. Uridinenucleotides are involved in activation and transfer of sugars toglycolipids and glycoproteins. Cytidine nucleotides are derived fromuridine nucleotides, and are crucial for synthesis of major membranephospholipid constituents like phosphatidylcholine, which receives itscholine moiety from cytidine diphosphocholine. In the case ofmitochondrial dysfunction (due to either mitochondrial DNA defects orany of the acquired or conditional deficits like exicitoxic or nitricoxide-mediated mitochondrial dysfunction described above) or otherconditions resulting in impaired pyrimidine synthesis, cellproliferation and axonal extension is impaired at crucial stages indevelopment of neuronal interconnections and circuits, resulting indelayed or arrested development of neuropsychological functions likelanguage, motor, social, executive function, and cognitive skills. Inautism for example, magnetic resonance spectroscopy measurements ofcerebral phosphate compounds indicates that there is globalundersynthesis of membranes and membrane precursors indicated by reducedlevels of uridine diphospho-sugars, and cytidine nucleotide derivativesinvolved in membrane synthesis (Minshew et al., Biological Psychiatry33:762-773, 1993).

Disorders characterized by developmental delay include Rett's Syndrome,pervasive developmental delay (or PDD-NOS: “pervasive developmentaldelay—not otherwise specified” to distinguish it from specificsubcategories like autism), autism, Asperger's Syndrome, and AttentionDeficit/Hyperactivity Disorder (ADHD), which is becoming recognized as adelay or lag in development of neural circuitry underlying executivefunctions.

Compounds and compositions of the invention are useful for treatingpatients with neurodevelopmental delays involving motor, language,executive function, and cognitive skills. Current treatments for suchconditions, e.g. ADHD, involve amphetamine-like stimulants that enhanceneurotransmission in some affected underdeveloped circuits, but suchagents, which may improve control of disruptive behaviors, do notimprove cognitive function, as they do not address underlying deficitsin the structure and interconnectedness of the implicated neuralcircuits.

Compounds and compositions of the invention are also useful in the caseof other delays or arrests of neurological and neuropsychologicaldevelopment in the nervous system and somatic development in non-neuraltissues like muscle and endocrine glands.

Treatment of Neurodegenerative Disorders

The two most significant severe neurodegenerative diseases associatedwith aging, Alzheimer's Disease (AD) and Parkinson's Disease (PD), bothinvolve mitochondrial dysfunction in their pathogenesis. Complex Ideficiencies in particular are frequently found not only in thenigrostriatal neurons that degenerate in Parkinson's disease, but alsoin peripheral tissues and cells like muscle and platelets of Parkinson'sDisease patients.

In Alzheimer's Disease, mitochondrial respiratory chain activity isoften depressed, especially Complex IV (Cytochrome c Oxidase). Moreover,mitochondrial respiratory function altogether is depressed as aconsequence of aging, further amplifying the deleterious sequelae ofadditional molecular lesions affecting respiratory chain function.

Other factors in addition to primary mitochondrial dysfunction underlieneurodegeneration in AD, PD, and related disorders. Excitotoxicstimulation and nitric oxide are implicated in both diseases, factorswhich both exacerbate mitochondrial respiratory chain deficits and whosedeleterious actions are exaggerated on a background of respiratory chaindysfunction.

Huntington's Disease also involves mitochondrial dysfunction in affectedbrain regions, with cooperative interactions of excitotoxic stimulationand mitochondrial dysfunction contributing to neuronal degeneration.

Compounds and compositions of the invention are useful for attenuatingprogression of age-related neurodegenerative disease including AD andPD.

Amyotrophic Lateral Sclerosis

One of the major genetic defects in patients with Amyotrophic LateralSclerosis (ALS; Lou Gehrig's Disease; progressive degeneration of motorneurons, skeletal muscle atrophy, inevitably leading to paralysis anddeath) is mutation or deficiency in Copper-Zinc Superoxide Dismutase(SOD1), an antioxidant enzyme. Mitochondria both produce and are primarytargets for reactive oxygen species. Inefficient transfer of electronsto oxygen in mitochondria is the most significant physiological sourceof free radicals in mammalian systems. Deficiencies in antioxidants orantioxidant enzymes can result in or exacerbate mitochondrialdegeneration. Mice transgenic for mutated SOD1 develop symptoms andpathology similar to those in human ALS. The development of the diseasein these animals has been shown to involve oxidative destruction ofmitochondria followed by functional decline of motor neurons and onsetof clinical symptoms (Kong and Xu, J. Neurosci. 18:3241-3250, 1998).Skeletal muscle from ALS patients has low mitochondrial Complex Iactivity (Wiedemann et al., J. Neurol. Sci 156:65-72, 1998).

Compounds, compositions, and methods of the invention are useful fortreatment of ALS, for reversing or slowing the progression of clinicalsymptoms.

Protection Against Ischemia and Hypoxia

Oxygen deficiency results in both direct inhibition of mitochondrialrespiratory chain activity by depriving cells of a terminal electronacceptor for Cytochrome c reoxidation at Complex IV, and indirectly,especially in the nervous system, via secondary post-anoxicexcitotoxicity and nitric oxide formation.

In conditions like cerebral anoxia, angina or sickle cell anemia crises,tissues are relatively hypoxic. In such cases, compounds of theinvention provide protection of affected tissues from deleteriouseffects of hypoxia, attenuate secondary delayed cell death, andaccelerate recovery from hypoxic tissue stress and injury.

Renal Tubular Acidosis

Acidosis due to renal dysfunction is often observed in patients withmitochondrial disease, whether the underlying respiratory chaindysfunction is congenital or induced by ischemia or cytotoxic agentslike cisplatin. Renal tubular acidosis often requires administration ofexogenous sodium bicarbonate to maintain blood and tissue pH.

In Example 3, administration of a compound of the invention caused animmediate reversal of renal tubular acidosis in a patient with a severeComplex I deficiency. Compounds and compositions of the invention areuseful for treating renal tubular acidosis and other forms of renaldysfunction caused by mitochondrial respiratory chain deficits.

Age-Related Neurodegeneration and Cognitive Decline

During normal aging, there is a progressive decline in mitochondrialrespiratory chain function. Beginning about age 40, there is anexponential rise in accumulation of mitochondrial DNA defects in humans,and a concurrent decline in nuclear-regulated elements of mitochondrialrespiratory activity.

de Grey (Bioessays, 19:161-167, 1998) discussed mechanisms underlyingthe observation that many mitochondrial DNA lesions have a selectionadvantage during mitochondrial turnover, especially in postmitoticcells. The proposed mechanism is that mitochondria with a defectiverespiratory chain produce less oxidative damage to themselves than domitochondria with intact functional respiratory chains (mitochondrialrespiration is the primary source of free radicals in the body).Therefore, normally-functioning mitochondria accumulate oxidative damageto membrane lipids more rapidly than do defective mitochondria, and aretherefore “tagged” for degradation by lysosomes. Since mitochondriawithin cells have a half life of about 10 days, a selection advantagecan result in rapid replacement of functional mitochondria with thosewith diminished respiratory activity, especially in slowly dividingcells. The net result is that once a mutation in a gene for amitochondrial protein that reduces oxidative damage to mitochondriaoccurs, such defective mitochondria will rapidly populate the cell,diminishing or eliminating its respiratory capabilities. Theaccumulation of such cells results in aging or degenerative disease atthe organismal level. This is consistent with the progressive mosaicappearance of cells with defective electron transport activity inmuscle, with cells almost devoid of Cytochrome c Oxidase (COX) activityinterspersed randomly amidst cells with normal activity, and a higherincidence of COX-negative cells in biopsies from older subjects. Theorganism, during aging, or in a variety of mitochondrial diseases, isthus faced with a situation in which irreplaceable postmitotic cells(e.g. neurons, skeletal and cardiac muscle) must be preserved and theirfunction maintained to a significant degree, in the face of aninexorable progressive decline in mitochondrial respiratory chainfunction. Neurons with dysfunctional mitochondria become progressivelymore sensitive to insults like excitotoxic injury. Mitochondrial failurecontributes to most degenerative diseases (especially neurodegeneration)that accompany aging.

Congenital mitochondrial diseases often involve early-onsetneurodegeneration similar in fundamental mechanism to disorders thatoccur during aging of people born with normal mitochondria. Thedemonstration disclosed in the Examples that compounds and compositionsof the invention are useful in treatment of congenital or early-onsetmitochondrial disease provides direct support for the utility ofcompounds and compositions of the invention for treatment of age-relatedtissue degeneration.

Compounds and compositions of the invention are useful for treating orattenuating cognitive decline and other degenerative consequences ofaging.

Mitochondria and Cancer Chemotherapy

Mitochondrial DNA is typically more vulnerable to damage than is nuclearDNA for several reasons:

-   -   1. Mitochondrial DNA has a less sophisticated repair system than        does nuclear DNA.    -   2. Virtually all of the mitochondrial DNA strands encode        important proteins, so that any defect will potentially affect        mitochondrial function. Nuclear DNA contains long regions that        do not encode proteins, wherein mutations or damage are        essentially inconsequential.    -   3. Defective mitochondria often have a selection advantage over        normal, active ones during proliferation and turnover.    -   4. Mitochondrial DNA is not protected by histones

Empirically, mitochondrial DNA damage is more extensive and persistslonger than nuclear DNA damage in cells subjected to oxidative stress orcancer chemotherapy agents like cisplatin due to both greatervulnerability and less efficient repair of mitochondrial DNA. Althoughmitochondrial DNA may be more sensitive to damage than nuclear DNA, itis relatively resistant, in some situations, to mutagenesis by chemicalcarcinogens. This is because mitochondria respond to some types ofmitochondrial DNA damage by destroying their defective genomes ratherthan attempting to repair them. This results in global mitochondrialdysfunction for a period after cytotoxic chemotherapy. Clinical use ofchemotherapy agents like cisplatin, mitomycin, and cytoxan is oftenaccompanied by debilitating “chemotherapy fatigue”, prolonged periods ofweakness and exercise intolerance which may persist even after recoveryfrom hematologic and gastrointestinal toxicities of such agents.

Compounds, compositions, and methods of the invention are useful fortreatment and prevention of side effects of cancer chemotherapy relatedto mitochondrial dysfunction. This use of pyrimidine nucleotideprecursors for attenuation of cancer chemotherapy side effects isconceptually and biochemically distinct from toxicity reduction ofcytotoxic anticancer pyrimidine analogs, which is mediated thoughbiochemical competition at the level of nucleotide antimetabolites.

Example 5 illustrates the protective effect of oral triacetyluridine inprotecting against taxol-induced neuropathy.

Furthermore, hepatic mitochondrial redox state is one contributor toappetite regulation. Cancer patients often display “early satiety”,contributing to anorexia, weight loss, and cachexia. Energy metabolismis often seriously disrupted in cancer patients, with energy-wastingfutile cycles of hyperactive tumor glycolysis producing circulatinglactate, which is converted by the liver back to glucose.Chemotherapy-induced mitochondrial injury further contributes tometabolic disruption.

As indicated in Example 2, treatment with a compound of the inventionresulted in improved appetite in a patient with mitochondrial disease.

Mitochondria and Ovarian Function

A crucial function of the ovary is to maintain integrity of themitochondrial genome in oocytes, since mitochondria passed on to a fetusare all derived from those present in oocytes at the time of conception.Deletions in mitochondrial DNA become detectable around the age ofmenopause, and are also associated with abnormal menstrual cycles. Sincecells cannot directly detect and respond to defects in mitochondrialDNA, but can only detect secondary effects that affect the cytoplasm,like impaired respiration, redox status, or deficits in pyrimidinesynthesis, such products of mitochondrial function participate as asignal for oocyte selection and follicular atresia, ultimatelytriggering menopause when maintenance of mitochondrial genomic fidelityand functional activity can no longer be guaranteed. This is analogousto apoptosis in cells with DNA damage, which undergo an active processof cellular suicide when genomic fidelity can no longer be achieved byrepair processes. Women with mitochondrial cytopathies affecting thegonads often undergo premature menopause or display primary cyclingabnormalities. Cytotoxic cancer chemotherapy often induces prematuremenopause, with a consequent increased risk of osteoporosis.Chemotherapy-induced amenorrhea is generally due to primary ovarianfailure. The incidence of chemotherapy-induced amenorrhea increases as afunction of age in premenopausal women receiving chemotherapy, pointingtoward mitochondrial involvement. Inhibitors of mitochondrialrespiration or protein synthesis inhibit hormone-induced ovulation, andfurthermore inhibit production of ovarian steroid hormones in responseto pituitary gonadotropins. Women with Downs syndrome typically undergomenopause prematurely, and also are subject to early onset ofAlzheimer-like dementia. Low activity of cytochrome oxidase isconsistently found in tissues of Downs patients and in late-onsetAlzheimer's Disease.

Appropriate support of mitochondrial function or compensation formitochondrial dysfunction therefore is useful for protecting againstage-related or chemotherapy-induced menopause or irregularities ofmenstrual cycling or ovulation. Compounds and compositions of theinvention, including also antioxidants and mitochondrial cofactors, areuseful for treating and preventing amenorrhea, irregular ovulation,menopause, or secondary consequences of menopause.

In Example 1, treatment with a compound of the invention resulted inshortening of the menstrual cycle. Since the patient was in a persistentluteal phase, her response indicates that the administered pyrimidinenucleotide precursor reversed hyporesponsiveness to pituitarygonadotropins, which were presumably elevated to compensate for theovarian hyporesponsiveness of mitochondrial origin.

Diagnosis of Mitochondrial Disease

The striking response of patients with mitochondrial disease toadministration of compounds of the invention indicates that a clinicalresponse to a pyrimidine nucleotide precursor administered according tothe methods of the subject invention has diagnostic utility to detectpossible mitochondrial disease. Molecular diagnosis of molecular lesionsunderlying mitochondrial dysfunction is difficult and costly, especiallywhen the defect is not one of the more common mutations or deletions ofmitochondrial DNA. Since the compounds and compositions of the inventionare very safe when administered in accord with the methods of thesubject invention, therapeutic challenge with a pyrimidine nucleotideprecursor is an important diagnostic probe for suspected mitochondrialdisease, especially when used in conjunction with tests for variousaspects of mitochondrial dysfunction.

E. Administration and Formulation of Compounds and Compositions of theInvention

In the case of all of the specific therapeutic targets for pyrimidinenucleotide precursor therapy of mitochondrial disease, compounds of theinvention are typically administered one to three times per day. Acylderivatives of uridine and cytidine are administered orally in doses of0.01 to 0.5 grams per kilogram of body weight per day, with variationswithin this range depending on the amount required for optimal clinicalbenefit. Generally, optimum doses are between 0.05 and 0.3 grams/kg/day,divided into two or three separate doses taken 6 to 12 hours apart.

In the case of patients unable to receive oral medications, compounds ofthe invention, especially uridine, cytidine, and orotate esters can beadministered, as required, by prolonged intravenous infusion, deliveringdaily doses of 0.01 to 0.5 grams/kg/day.

The pharmacologically active compounds optionally are combined withsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds. Theseare administered as tablets, suspensions, solutions, dragees, capsules,or suppositories. The compositions are administered for example orally,rectally, vaginally, or released through the buccal pouch of the mouth,and may be applied in solution form by injection, orally or by topicaladministration. The compositions may contain from about 0.1 to 99percent, preferably from about 50 to 90 percent of the activecompound(s), together with the excipient(s).

For parenteral administration by injection or intravenous infusion, theactive compounds are suspended or dissolved in aqueous medium such assterile water or saline solution. Injectable solutions or suspensionsoptionally contain a surfactant agent such as polyoxyethylenesorbitanesters, sorbitan esters, polyoxyethylene ethers, or solubilizing agentslike propylene glycol or ethanol. The solution typically contains 0.01to 5% of the active compounds.

Suitable excipients include fillers such as sugars, for example lactose,sucrose, mannitol or sorbitol, cellulose preparations and/or calciumphosphates, for example tricalcium phosphate or calcium hydrogenphosphate, as well as binders such as starch paste, using, for example,maize starch, wheat starch, rice starch or potato starch, gelatin,tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodiumcarboxymethyl cellulose and/or polyvinyl pyrrolidone.

Auxiliaries include flow-regulating agents and lubricants, for example,silica, talc, stearic acid or salts thereof, such as magnesium stearateor calcium stearate and/or polyethylene glycol. Dragee cores areprovided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated sugar solutions are used,which optionally contain gum arabic, talc, polyvinyl pyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvents or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate are used. Dyestuffs or pigmentsare optionally added to the tablets or dragee coatings, for example, foridentification or in order to characterize different compound doses.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral useare obtained by combining the active compound(s) with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Other pharmaceutical preparations which are useful for oral deliveryinclude push-fit capsules made of gelatin, as well as soft-sealedcapsules made of gelatin and a plasticizer such as glycerol or sorbitol.The push-fit capsules contain the active compound(s) in the form ofgranules which optionally are mixed with fillers such as lactose,binders such as starches and/or lubricants such as talc or magnesiumstearate, and, optionally stabilizers. In soft capsules, the activecompounds are preferably dissolved or suspended in suitable liquids suchas fatty oils, liquid paraffin, or polyethylene glycols. In addition,stabilizers optionally are added.

Pharmaceutical preparations which are used rectally include, forexample, suppositories which consist of a combination of activecompounds with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, paraffin hydrocarbons,polyethylene glycols or higher alkanols. In addition, gelatin rectalcapsules which consist of a combination of the active compounds with abase are useful. Base materials include, for example, liquidtriglycerides, polyethylene glycols, or paraffin hydrocarbons. Inanother embodiment of the invention, an enema formulation is used, whichoptionally contains viscosity-increasing excipients likemethylcellulose, hydroxypropylmethylcellulose, carboxymethycellulose,carbopol, glycerine polyacrylates, or other hydrogels.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water soluble form, for example,water soluble salts.

In addition, suspensions of the active compounds as appropriate in oilyinjection suspensions are administered. Suitable lipophilic solvents orvehicles include fatty oils, for example, sesame oil, or synthetic fattyacid esters, for example, ethyl oleate or triglycerides. Aqueousinjection suspensions optionally include substances which increase theviscosity of the suspension which include, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspensionoptionally contains stabilizers.

F. Synthesis of the Compounds of the Invention

Acyl derivatives of cytidine and uridine are synthesized typically byacylation methods involving reaction of acid chlorides or acidanhydrides with cytidine or uridine.

The synthesis of 2′,3′,5′-tri-O-pyruvyluridine is shown in Example 6.

The following examples are illustrative, but not limiting of the methodsand compositions of the present invention. Other suitable modificationsand adaptations of a variety of conditions and parameters normallyencountered in clinical therapy which are obvious to those skilled inthe art are within the spirit and scope of this invention.

EXAMPLES Example 1 Treatment of a Multisystem Mitochondrial Disorderwith Triacetyluridine

A 29 year old woman with a partial Complex I deficiency, and whose sonwas diagnosed with mitochondrial disease leading to stroke-likeepisodes, ataxia, and encephalopathy, presented with a multisystemmitochondrial disorder. Signs and symptoms included hemiplegic/aphasicmigraines, grand-mal seizures, neurogenic bowel and bladder dysfunction,requiring catheterization approximately four times per day, dysphagia,autonomic and peripheral polyneuropathy producing painful paresthesias,tachycardia/bradycardia syndrome, and poor functional capacity withinability to climb a flight of stairs without stopping to rest, anddeclining cognitive performance with episodes of clouded sensorium andpoor memory lasting hours to days.

After beginning treatment with 0.05 mg/kg/day of oral triacetyluridine,and for a duration of at least 6 months, this patient has not hadseizures or migraines; her paresthesias related to peripheral neuropathyhave resolved. She is able to void spontaneously on most days, requiringcatheterization only once or twice per week. After 6 weeks of treatmentwith triacetyluridine, this patient was able to walk a full mile, whichshe has been unable to do for the past two years because of inadequatefunctional capacity. Her episodes of bradycardia during sleep andtachycardia during exertion have reduced in frequency; prior totreatment, tachycardia with a heart rate greater than 140 bpm occurredupon simple rise to stand, and after 6 weeks of triacetyluridine,tachycardia occurred only on hills and stairs. Her sensorium has clearedand memory deficits have improved markedly.

During treatment, this patients' menstrual cycles shortened from 4 weeksto two weeks, and she displayed a persistent luteal phase as evaluatedby estradiol, progesterone, FSH and LH measurements. After severalmonths, her cycle normalized to 4 weeks.

This patient demonstrates important features of the subject invention,in that 1) the compound of the invention caused improvements invirtually all features of a complex multisystem disease related tomitochondrial dysfunction in a variety of tissues, and that 2) compoundsof the invention are unexpectedly useful for treating disease conditionsrelated to a partial Complex I deficiency, which affects a portion ofthe mitochondrial respiratory chain that is outside of the sequence ofelectron transfers directly involved in de novo pyrimidine biosynthesis.

The transient shortening of this patient's menstrual cycle isinterpreted as an improvement of ovarian function caused bytriacetyluridine in the face of excessive hormonal stimulation by whichthe neuroendocrine system was attempting to compensate for ovariandysfunction. Feedback between the ovaries and the hypothalamus led togradual normalization of cycle time.

Example 2 Treatment of Refractory Epilepsy

An 11 year old boy had refractory epilepsy since age 4.5, apparently dueto a multiple mitochondrial DNA deletion syndrome. In December 1997, hiscondition deteriorated, including 2 admissions to an intensive care unitfor crescendo epilepsy. Even with aggressive regimens of standardanticonvulsive therapy, this patient was having 8 to 10 grand-malseizures per night, leaving him unable to attend school regularly orparticipate in sports activities. He also developed upper lipautomaticity.

In the first three days after beginning treatment with oraltriacetyluridine (initially at a dose of 0.05 g/kg/day, andincrementally increased to 0.1 and then 0.24 g/kg/day over the course ofseveral weeks), there were no seizures, and involuntary lip movementsceased. There has subsequently been some recurrence of seizuresespecially during episodes of infection, though at a much lowerfrequency than prior to treatment with triacetyluridine. This patienthas been able to return to school and resume active participation insports. His appetite, cognitive function, and fine motor coordinationhave improved during therapy, resulting in improved academic performanceand in outstanding performance in sports activities like baseball.

Example 3 Treatment of Renal Tubular Acidosis

A 2 year-old girl, with Leigh's Syndrome (subacute necrotizingencephalopathy) associated with severe Complex I deficiency, displayedrenal tubular acidosis requiring intravenous administration of 25 mEqper day of sodium bicarbonate. Within several hours after beginningintragastric treatment with triacetyluridine at 0.1 g/mg/day, her renaltubular acidosis resolved and supplementary bicarbonate was no longerrequired to normalize blood pH. Triacetyluridine also resulted in rapidnormalization of elevated circulating amino acid concentrations, andmaintained lactic acid at low levels after withdrawal of dichloroacetatetreatment, which was previously required to prevent lactic acidosis.

Example 4 Treatment of Developmental Delay

A 4.5 year-old girl with epilepsy, ataxia, language delay, and fatintolerance, and dicarboxylic aciduria was treated with triacetyluridineat a daily dose of 0.1 to 0.3 g/kg/day. Such treatment resulted in a 50%decline in seizure frequency, improvement of ataxia and motorcoordination, restoration of dietary fat tolerance, and accelerateddevelopment of expressive language capabilities.

Example 5 Prevention of Taxol-Induced Neuropathy

Peripheral neuropathy is a frequent, and often dose-limiting, sideeffect of important anticancer agents like cisplatin and taxol. In thecase of taxol, sensory neuropathy occurs several days afteradministration. Taxol's mechanism of action involves stabilization ofmicrotubules, which is useful for treating cancers, but is deleteriousto peripheral neurons. Microtubule stabilization impairs axonaltransport of cellular components. Mitochondria shuttle between the cellbody and terminals of neurons, so that the expression of mitochondrialrespiratory chain components can be regulated by nuclear transcriptionfactors. During inhibition of mitochondrial shuttling, mitochondriadistant from the nucleus undergo decline in expression of respiratorychain subunits encoded by the mitochondrial genome, due to inadequateexposure to mDNA transcription factors, resulting in regional neuronalenergy failure and other consequences of mitochondrial dysfunction.

Two groups of 10 mice each were treated with taxol, 21.6 mg/kg/day for 6consecutive days by intraperitoneal injection. An additional group of 10mice received injections of vehicle alone. One of the groups oftaxol-treated mice received oral triacetyluridine, 4000 mg/kg b.i.d.Nine days after the initiation of taxol treatments, nociceptive sensorydeficits were tested by determining tail-flick latency after exposure ofthe tip of the tail to focused thermal radiation with an infrared heatlamp. In this system, delays in the tail-flick response to radiant heatcorrelate with sensory nerve deficits. Group: Tail flick latency Control(no taxol) 10.8 ± 0.5 seconds Taxol 16.0 ± 3.1 seconds Taxol +triacetyluridine 11.9 ± 0.7 seconds

Taxol treatment impaired responses to painful stimuli as an index oftoxic sensory neuropathy. Oral triacetyluridine treatment significantlyattenuated taxol-induced alterations in tail-flick latency.

Example 6 Synthesis of Uridine Pyruvate

A. The preparation of pyruvyl chloride was accomplished by the reactionof alpha, alpha-dichloromethyl methyl ether and pyruvic acid using theprocedure of Ottenheum and Man (Synthesis, 1975, p. 163).

B. Uridine (3.0 g, 12 mmol) was dried by toluene azeotrope under vacuum(3×), and then dissolved in DMF (20 mL) and pyridine (20 mL). Theresultant solution was cooled to −10 degrees C. and 6.0 mL of pyruvylchloride (produced in step A above) was added dropwise. The reactionmixture was stirred at room temperature under argon for 24 hours.Analysis by TLC (5% MeOH/CH₂Cl₂) showed the consumption of uridine. Thereaction mixture was evaporated to dryness and partitioned betweenCH₂Cl₂ and aqueous sodium bicarbonate. The organic layer was washed withwater, aqueous HCl (pH 3.0), and water; dried over sodium sulfate;concentrated; and purified using flash chromatography (silica gel, 5%MeOH/C₂Cl₂) to yield 1.4 g of uridine pyruvate, or2′,3′,5′-tri-O-pyruvyluridine.

While the present invention has been described in terms of preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations and modificationswhich come within the scope of the invention as claimed.

1. A method for treating or preventing pathophysiological consequencesof mitochondrial respiratory chain dysfunction in a mammal comprisingadministering to said mammal in need of such treatment or prevention aneffective amount of a pyrimidine nucleotide precursor.
 2. A method as inclaim 1 wherein said respiratory chain dysfunction is caused by amutation, deletion, or rearrangement of mitochondrial DNA.
 3. A methodas in claim 1 wherein said respiratory chain dysfunction is caused bydefective nuclear-encoded protein components of the mitochondrialrespiratory chain.
 4. A method as in claim 1 wherein said respiratorychain dysfunction is caused by aging.
 5. A method as in claim 1 whereinsaid respiratory chain dysfunction is caused by administration ofcytotoxic cancer chemotherapy agents to said mammal.
 6. A method as inclaim 1 wherein said respiratory chain dysfunction is a deficit inmitochondrial Complex I activity.
 7. A method as in claim 1 wherein saidrespiratory chain dysfunction is a deficit in mitochondrial Complex IIactivity.
 8. A method as in claim 1 wherein said respiratory chaindysfunction is a deficit in mitochondrial Complex III activity.
 9. Amethod as in claim 1 wherein said respiratory chain dysfunction is adeficit in mitochondrial Complex IV activity.
 10. A method as in claim 1wherein said respiratory chain dysfunction is a deficit in mitochondrialComplex V activity.
 11. A method as in claim 1 wherein said pyrimidinenucleotide precursor is selected from the group consisting of uridine,cytidine, an acyl derivative of uridine, an acyl derivative of cytidine,orotic acid, an alcohol ester of orotic acid, or a pharmaceuticallyacceptable salt thereof.
 12. A method as in claim 11 wherein saidpyrimidine nucleotide precursor is an acyl derivative of cytidine.
 13. Amethod as in claim 11 wherein said pyrimidine nucleotide precursor is anacyl derivative of uridine.
 14. A method as in claim 11 wherein saidacyl derivative of uridine is 2′, 3′,5′-tri-O-acetyluridine
 15. A methodas in claim 11 wherein said acyl derivative of uridine is2′,3′,5′-tri-O-pyruvyluridine.
 16. A method as in claim 11 wherein thealcohol substitutent of said alcohol ester of orotic acid is ethanol.17. A method as in claim 11 wherein said pyrimidine nucleotide precursoris cytidine diphosphocholine.
 18. A method as in claim 11 wherein saidpyrimidine nucleotide precursor is administered orally.
 19. A method asin claim 11 wherein said pyrimidine nucleotide precursor is administeredin a dose of 0.01 to 1 gram per kilogram of bodyweight per day.
 20. Amethod as in claim 1 wherein said pathophysiological consequence ofmitochondrial respiratory chain dysfunction is a congenitalmitochondrial disease.
 21. A method as in claim 20 wherein saidcongenital mitochondrial disease is selected from the group consistingof MELAS, LHON, MERRF, NARP, PEO, Leigh's Disease, and Kearns-SayresSyndrome.
 22. A method as in claim 1 wherein said pathophysiologicalconsequence of mitochondrial respiratory chain dysfunction is aneurodegenerative disease.
 23. A method as in claim 22 wherein saidneurodegenerative disorder is Alzheimer's Disease.
 24. A method as inclaim 22 wherein said neurodegenerative disorder is Parkinson's disease.25. A method as in claim 22 wherein said neurodegenerative disorder isHuntington's Disease.
 26. A method as in claim 22 wherein saidneurodegenerative disorder is age-related decline in cognitive function.27. A method as in claim 1 wherein said pathophysiological consequenceof mitochondrial respiratory chain dysfunction is a neuromusculardegenerative disease.
 28. A method as in claim 27 wherein saidneuromuscular degenerative disease is selected from the group consistingof muscular dystrophy, myotonic dystrophy, chronic fatigue syndrome, andFriedreich's Ataxia.
 29. A method as in claim 1 wherein saidpathophysiological consequence of mitochondrial respiratory chaindysfunction is developmental delay in cognitive, motor, language,executive function, or social skills.
 30. A method as in claim 1 whereinsaid pathophysiological consequence of mitochondrial respiratory chaindysfunction is selected from the group consisting of epilepsy,peripheral neuropathy, optic neuropathy, autonomic neuropathy,neurogenic bowel dysfunction, sensorineural deafness, neurogenic bladderdysfunction, migraine, and ataxia.
 31. A method as in claim 1 whereinsaid pathophysiological consequence of mitochondrial respiratory chaindysfunction is selected from the group consisting of renal tubularacidosis, dilating cardiomyopathy, hepatic failure, and lactic acidemia.32. A method for preventing death or functional decline of post-mitoticcells in a mammal due to mitochondrial respiratory chain dysfunctioncomprising administration of an effective amount of a pyrimidinenucleotide precursor.
 33. A method as in claim 32 wherein saidpost-mitotic cells are neurons.
 34. A method as in claim 32 wherein saidpost-mitotic cells are skeletal muscle cells.
 35. A method as in claim32 wherein said post-mitotic cells are cardiomyocytes.
 36. A method fortreating developmental delay in cognitive, motor, language, executivefunction, or social skills in a mammal comprising administration of aneffective amount of a pyrimidine nucleotide precursor.
 37. A method asin claim 36 wherein said developmental delay is pervasive developmentaldelay or PDD-NOS.
 38. A method as in claim 36 wherein said developmentaldelay is Attention Deficit/Hyperactivity Disorder.
 39. A method as inclaim 36 wherein said developmental delay is Rett's Syndrome.
 40. Amethod as in claim 36 wherein said developmental delay is autism.
 41. Amethod for reducing side effects of cytotoxic cancer chemotherapy agentsby administering a pyrimidine nucleotide precursor, where said cytotoxicchemotherapy agent is not a pyrimidine nucleoside analog.
 42. A methodas in claim 41 wherein said side effects of cytotoxic cancerchemotherapy are selected from the group consisting of peripheralneuropathy, chemotherapy-induced menopause, chemotherapy-associatedfatigue, and depressed appetite.
 43. A method for diagnosingmitochondrial disease by administering a pyrimidine nucleotide precursorand assessing clinical improvement in signs and symptoms in a mammal.44. A compound selected from the group consisting of2′,3′,5′-tri-O-pyruvyluridine, 2′,3′-di-O-pyruvyluridine,2′,5′-di-O-pyruvyluridine, 3′,5′-di-O-pyruvyluridine,2′-O-pyruvyluridine, 3′-O-pyruvyluridine, and 5′-O-pyruvyluridine.
 45. Apharmaceutical composition comprising: (a) a pyrimidine nucleotideprecursor or a pharmaceutically acceptable salt thereof, and (b) pyruvicacid, a pharmaceutically acceptable salt thereof, or a pyruvic acidester.
 46. A method as in claim 1 further comprising administeringpyruvic acid, a pharmaceutically acceptable salt thereof, or a pyruvicacid ester.